CRISPR/Cas9载体优化及CHD1L条件性敲除肝癌细胞系的构建

The CRISPR/Cas9 system optimization and construction of CHD1L conditional knockout liver cancer cell lines

目的利用优化的pLV-Tet3G-CRISPR/Cas9载体系统构建条件性敲除CHD1L的QGY-7703肝癌细胞系,验证敲除效果及其对细胞生物学的影响,为研究CHD1L促肿瘤细胞恶性表型机制提供重要的细胞模型。方法用点突变方法优化pLV-Tet3G-Cas9载体以降低其脱靶效应,进而将Cas9改造成eSpCas9;其次,通过筛选获得稳定表达eSpCas9的QGY-7703细胞株;将mCherry基因插入载体pLVXhU6-SgRNA中,获得携带mCherry荧光基因的pLVX-mCherry-hU6-SgRNA载体;设计和筛选特异性靶向CHD1L的SgRNA序列,用重叠PCR方法获得hU6-CHD1L-SgRNA片段,筛选具有CHD1L切割活性的靶点,随后,将其克隆到pLVX-mCherry-hU6-SgRNA载体中;用293FT细胞进行病毒包装,获得慢病毒颗粒;转染7703eSpCas9细胞株,利用Western blot验证Dox诱导下的CHD1L敲除效果,划痕和Transwell实验检测Dox诱导的CHD1L敲除对肝癌细胞生物学功能的影响。结果 pLV-Tet3G-Cas9载体Cas9成功优化为eSpCas9序列;成功构建pLVX-mCherry-hU6-CHD1L-SgRNA载体;通过转染及筛选,获得Dox诱导的CHD1L敲除QGY-7703肝癌细胞系;细胞实验显示Dox可诱导eSpCas9表达,靶向性切割CHD1L,抑制QGY-7703细胞的迁移侵袭。结论成功构建Dox诱导的CHD1L敲除肝癌细胞株,此载体系统可为靶向肿瘤特异性基因研究提供细胞模型。

Objective To verify the effect of CHD1 L QGY-7703 hepatoma cell line knockout and the effect on the cell biological functions and then to provide important cells models for studying the role of CHD1 L in the development of mechanism of malignant phenotype in cancer cell by using the optimized p LVTet3 G-CRISPR/Cas9 technology to construct a QGY-7703 hepatoma cell line. Methods p LV-Tet3 G-Cas9 vector was optimized by point mutation technique to reduce its off-target effect and increase specificity. Cas9 was transformed into e Sp Cas9. Then, QGY-7703 cell line stably expressing e Sp Cas9 was obtained by screening. p LVX-m Cherry-h U6-Sg RNA vector carrying m Cherry fluorescent gene was obtained by inserting m Cherry gene into p LVX-h U6-Sg RNA vector. The sg RNA sequence targeting the CHD1 L gene was designed and screened, and the targeting sequence of Hu6-CHD1 L-sg RNA was obtained by overlapping PCR method.Cloning the site with cleavage activity into the p LVX-m Cherry-h U6-Sg RNA vector. The vector was packaged as lentivirus by lentiviral packaging method and transfected into 7703 e Sp Cas9 cell line. The CHD1 L knockout induced by Dox was verified by Western blot. The effects of Dox-induced CHD1 L knockout on the biological function of hepatocellular carcinoma cells were detected by Wound Healing and Transwell assay.Results In this experiment, vector Cas9 of p LV-Tet3 G-Cas9 was successfully optimized as sequence e Sp Cas9. p LVX-m Cherry-h U6-CHD1 L-Sg RNA vector was constructed. The QGY-7703 liver cancer cell line that conditional induction CHD1 L gene knocking out was successfully constructed. Cell function experiments showed that the expression of e Sp Cas9 can be induced by Dox, the migration and invasion ability of these hepatoma cells were significantly decreased after knockout of CHD1 L. Conclusions This study successfully constructed a conditional CHD1 L knockout QGY-7703 liver cancer cell line. This model can be used as an effective tool to study the function and mechanism of specific genes targeting tumors.